Abstract: Structure, Function, and Disruption of Microbial Amyloid Assembly and Biofilm Formation Biofilm bacteria are wreaking havoc in the clinic. Our current arsenal of antibiotics is comprised of variations on the single theme of cell killing, i.e. targeting processes essential to bacterial viability, yet biofilms exhibit reduced sensitivity to conventional antibiotics and host defenses, and have emerged as virulence hallmarks of serious and persistent infectious diseases, including cystic fibrosis, urinary tract infection, endocarditis, and catheter infections. Improved biofilm models are crucial to understanding function and driving the design of new anti-infectives. In this New Innovator Award application I present an unconventional research plan designed to enable and deliver breakthrough discoveries needed to transform biofilm descriptors from vague terms like "glue" and "slime" to quantitative descriptions based on chemical composition and molecular architecture. I achieve this by integrating new whole-cell solid-state NMR strategies with chemical biology, microscopy, and biochemical techniques. From a "systems perspective," carbon and nitrogen NMR spectra of intact biofilms will permit a sum-of-all-parts analysis, while selective recoupling measurements, together with steady-state and transient biosynthetic labeling, will assign unique molecular signatures to permit biofilm profiling as a function of time and organism. Bacterial amyloids are prevalent in biofilms among diverse phyla and contribute to biofilm formation and virulence by uropathogenic E. coli. We will dissect in atomic-level detail the molecular basis of amyloid biogenesis. We will then establish how bacteria use these structures as building blocks in the construction of biofilm architectures. We will also be engaged in identifying compounds that interfere with assembly processes and will examine the nature of disruption to improve our understanding of the multi-protein amyloid machinery and biofilm manufacturing in E. coli vis-[unreadable]-vis chemical genetics. In a broader context, results of this project over the next five years will establish an experimental basis for quantitatively characterizing heterogeneous, insoluble, noncrystalline assemblies that contribute to human infectious diseases. Public Health Relevance: This era may come to be remembered as one in which infectious diseases made a dramatic worldwide resurgence, owing to the challenge of treating persistent biofilmassociated infectious diseases, the increasing rise of antibiotic resistance, and the dwindling number of candidate antibiotics in the drug development pipeline. We propose an unconventional and innovative approach that will enable transformative discoveries to understand at a molecular and atomic level how bacteria assemble complex heteropolymeric extracellular structures, including functional amyloid fibers, and how bacteria use these building blocks to construct organized biofilm architectures. We will also be engaged in identifying small molecules to interfere with assembly processes to drive the development of new anti-amyloid, anti-biofilm, and anti-virulence therapeutics.